Throughout this application, citations for various publications are provided within parentheses in the text. The disclosures of these publications are hereby incorporated in their entirety by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The function of carbohydrates as structural materials and as energy storage units in biological systems is well recognized. By contrast, the role of carbohydrates as signaling molecules in the context of biological processes has only recently been appreciated. (M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Singhal, S. Hakomori, J. C. Paulson, Science 1990, 250, 1130; M. J. Polley, M. L. Phillips, E. Wagner, E. Nudelman, A. K. Singhal, S. Hakomori, J. C. Paulson, Proc. Natl. Acad. Sci. USA 1991, 88, 6224: T. Taki, Y. Hirabayashi, H. Ishikawa, S. Kon, Y. Tanaka, M. Matsumoto, J. Biol. Chem. 1986, 261, 3075; Y. Hirabayashi, A. Hyogo, T. Nakao, K. Tsuchiya, Y. Suzuki, M. Matsumoto, K. Kon, S. Ando, ibid., 1990, 265, 8144; O. Hindsgaul, T. Norberg, J. Le Pendu, R. U. Lemieux, Carbohydr. Res. 1982, 109, 109; U. Spohr, R. U. Lemieux, ibid., 1988, 174, 211).
The elucidation of the scope of carbohydrate involvement in mediating cellular interaction is an important area of inquiry in contemporary biomedical research. The carbohydrate molecules, carrying detailed structural information, tend to exist as glycoconjugates (cf. glycoproteins and glycolipids) rather than as free entities. Given the complexities often associated with isolating the conjugates in homogeneous form, and the difficulties in retrieving intact carbohydrates from these naturally occurring conjugates, the opportunities for synthesis are apparent. (For recent reviews of glycosylation see: Paulsen, H., Agnew. Chemie Int. Ed. Engl. 1982, 21, 155; Schmidt, R. R., Angew. Chemie Int. Ed. Engl. 1986, 25, 212; Schmidt, R. R., Comprehensive Organic Synthesis, Vol. 6, Chapter 1 (2) Pergamom Press, Oxford, 1991; Schmidt, R. R., Carbohydrates, Synthetic Methods and Applications in Medicinal Chemistry, Part I, Chapter 4, VCH Publishers, Weinheim, New York, 1992. For pioneering work on the use of glycals as glycosyl donors in glycoside synthesis see Lemieux, R. U., Can. J. Chem. 1964, 42, 1417; Lemieux, R. U., Faser-Reid, B. Can. J. Chem. 1965, 42, 1460; Lemieux, R. U., Morgan, A. R., Can. J. Chem. 1965, 43, 2190; Thiem, J., Karl, H., Schwentner, J., Synthesis 1978, 696; Thiem. J. Ossowski, P., Carbohydr. Chem. 1984, 3, 287; Thiem, J., Prahst, A., Wendt, T. Liebigs Am. Chem. 1986, 1044; Thiem, J. in Trends in Synthetic Carbohydrate Chemistry, Horton, D., Hawkins, L. D., McGarvvey, G. L. (Eds.), ACS Symposium Series #386, American Chemical Society, Washington, D.C., 1989, Chapter 8.)
The carbohydrate domains of the blood group substances, contained in both glycoproteins and glycolipids, are distributed in erythrocytes, epithelial cells and in various secretions. The early focus on these systems centered on their central role in determining blood group specificities. (R. R. Race, R. Sanger, Blood Groups in Man, 6th ed., Blackwell, Oxford, 1975) However, it is recognized that such determinants are broadly implicated in cell adhesion and binding phenomena. (For example, see M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Singhal, S. Hakomori, J. C. Paulson, Science 1990, 250, 1130.) Moreover, ensembles related to the blood group substances in conjugated form are encountered as markers for the onset of various tumors. (K. O. Lloyd, Am. J. Clinical Path. 1987, 87, 129; K. O. Lloyd Cancer Biol. 1991, 2, 421) Carbohydrate-based tumor antigenic factors might find applications at the diagnostic level, as resources in drug delivery, or, ideally, in immunotherapy. (T. Toyokuni, B. Dean, S. Cai, D. Boivin, S. Hakomori A. K. Singhal, J. Am. Chem Soc. 1994, 116, 395; G. Dranoff, E. Jaffee, A. Lazenby, P. Golumbek, H. Levitsky, K. Brose, V. Jackson, H. Hamada, D. Paardoll, R. Mulligan, Proc. Natl. Acad. Sci. USA 1993, 90, 3539; M-H. Tao, R. Levy Nature 1993, 362, 755; d) T. Boon, Int. J. Cancer 1993, 54, 177; P. O. Livingston, Curr. Opin. Immunol. 1992, 4, 624; S. Hakomori, Annu. Rev. Immunol. 1984, 2, 103; K. Shigeta, Y. Ito, T. Ogawa, Y. Kirihata, S. Hakomori, R. Kannagi, J. Biol. Chem. 1987, 262, 1358)
The present invention provides new strategies and protocols for oligosaccharide synthesis. The object is to simplify such constructions such that relatively complex domains can be assembled with high stereospecifity. Major advances in glycoconjugate synthesis require the attainment of high degrees of convergence and relief from the burdens associated with the manipulation of blocking groups. Another requirement is that of delivering the carbohydrate determinant with appropriate provision for conjugation to carrier proteins or lipids. (M. A. Bernstein, L. D. Hall, Carbohydr. Res. 1980, 78, Cl; R. U. Lemieux, Chem. Soc. Rev. 1978, 7, 423; R. U. Lemieux, D. R. Bundle, D. A. Baker, J. Am. Chem. Soc. 1975, 97, 4076) This is a critical condition if the synthetically derived carbohydrates are to be incorporated into carriers suitable for biological application.
The present invention shows how the use of glycals both as glycosyl donors and as glycosyl acceptors can be exploited to accomplish such ends in the context of a straightforward synthesis of the Le.sup.y (type II) system. The Le.sup.y hapten was first isolated from a blood group glycoprotein in 1966 by Kabat and Lloyd. (K. O. Lloyd, E. A. Kabat, E. J. Layug, F. Gruezo, Biochem. 1966, 5, 1489) Subsequently, Potapov and coworkers (M. I. Potapov, Probl. Hematol. Blood Transfus. (USSR) 1970, 15, 45) discovered an antibody to this carbohydrate antigen. Interest in the Le.sup.y antigen stems from the existence of a number of glycoproteins and glycolipids which contain this substructure and are associated with human colonic adenocarcinoma (T. Kaizu, S. B. Levery, E. Nudelman, R. E. Stenkamp, S. Hakomori, J. Biol. Chem. 1986, 261, 11254) as well as human liver adenocarcinoma. (S. B. Levery, E. Nudelman, N. H. Anderson, S. Hakomori, Carbohydr. Res. 1986, 151, 311) Both Le.sup.y and Le.sup.x antigenic structures are of general interest due to the preponderance of highly fucosylated polylactosamine glycolipids that are found to accumulate in other human carcinomas. (S. Hakomori, Annu. Rev. Immunol. 1984, 2, 103; S. Hakomori, E. Nudelman, S. B. Levery, R. Kannagi, J. Biol. Chem, 1984, 259, 4672; Y. Fukushi, S. Kakomori, E. Nudelman. N. Cochran, ibid. 1984, 259, 4681; Y. Fukushi, E. Nudelman, S. B. Levery, S. Hakomori, H. Rauvala, ibid. 1984, 259, 10511) The method of synthesis disclosed herein provides for the determinant to be insulated from the conjugation device by a carbohydrate spacer module which can in principle be broadly varied. Through appropriate insulation, the likelihood that the protein or lipid carrier might distort the recognition property of the determinant is thus minimized.
Also disclosed herein is the construction of the Lewis.sup.b determinant. (For alternative methods for synthesizing Le.sup.2 oligosaccharides, see: S. S. Rana, J. J. Barlow, K. L. Matta, Carbohydr. Res. 1981, 96, 231; U. Spohr, R. U. Lemieux, Carbohydr. Res. 1988, 174, 211) The present invention provides a method of equipping the reducing end of the antigen with a suitable device for subsequent attachment to a carrier protein. An intervening spacer element (lactose) is incorporated to insulate the recognition domain from the bioconjugation module.